Oncolytic herpes simplex virus (oHSV) is one of the most promising candidates for glioblastoma (GBM) therapy: is inherently neurotropic and can replicate in neoplastic cells and mediate robust oncolytic effects while sparing normal cells. Although phase I and Ib oHSV clinical trials conducted to date for GBM have shown signs of anti- tumor activity, clinical response rates have been sub-optimal, primarily due to tumor heterogeneity and oHSV delivery issues in the tumor resection cavity post-surgery. Based on our recent finding that oHSV susceptibility varies among tumor lines, we have engineered an armed oHSV mutant encoding S-TRAIL (secretable tumor necrosis factor-related apoptosis-inducing ligand) (oHSV-TRAIL) and shown killing of oHSV resistant GBM lines in a mechanism based manner. In our recently published studies, we have shown that human mesenchymal stem cell (MSC) loaded oHSV have a significantly better therapeutic efficacy than the purified oHSV in a mouse GBM model. These results although promising, have raised fundamental questions on how to specifically target heterogeneous and invading GBM tumor residues after resection of primary GBM as well as recurrent GBMs that have undergone the current standard of care? In the proposed studies, we will utilize patient derived primary GBM stem cells (GSC: invasive and nodular), pharmacological inhibitors and genetic gain/loss of function constructs to elucidate the molecular mechanism of MSC loaded oHSV-TRAIL mediated GBM killing. We hypothesize that MSC-oHSV-TRAIL will specifically kill a broad spectrum of GSC by targeting both cell proliferation and death pathways. The delivery, spread and therapeutic efficacy of MSC loaded oHSV-TRAIL will be tested in nodular and highly invasive mouse tumor models of resection generated from primary GSC. Based on our recent findings that synthetic extracellular matrix (sECM) encapsulation of stem cells is necessary to prevent rapid wash- out of stem cells post- transplantation in the tumor resection cavity, we will encapsulate MSC loaded oHSV-TRAIL into sECM and evaluate their fate and efficacy in tumor resection cavity generated from primary GSC lines. We hypothesize that sECM-MSC-oHSV-TRAIL will effectively target residual tumor cells in the resection cavity and also track and kill invasive tumor cells. Finally, the responses of recurrent GSC, obtained from GBM patients that are resistant to standard therapies, to MSC-oHSV-TRAIL will be assessed in vitro and the status of proteins involved in survival and apoptosis pathways will be analyzed. Intracerebral mouse models of invasive and nodular recurrent GBM will be generated and the efficacy of intracarotid artery (ICA) delivered MSC-oHSV-TRAIL will be tested. We hypothesize that MSC loading will protect virus from neutralization and MSC will efficiently extravasate and home to the tumor sites resulting in specific killing of recurrent GSC. Fluorescent and bioluminescent imaging (BLI) markers will be genetically incorporated into oHSV and GSC to follow fate of oHSV, GBM cell invasion and oHSV efficacy by real time in vivo imaging. The proposed studies are likely to unravel the mechanism-based, targeted stem cell mediated therapies for malignant GBMs which can be readily translated into clinics.